Rift Valley fever virus (RVFV) is a prototypical virus of the genus Phlebovirus, in the family Bunyaviridae, and belongs to the NIAID Category A list pathogens and the CDC list of potential bioterrorism agents. Rift Valley fever is an endemic disease of sub-Saharan Africa that can emerge in explosive mosquito-borne epidemics, decimating herds of sheep and cattle and resulting in enormous economic losses;humans are infected concurrently, resulting in hemorrhagic fever, encephalitis, and retinal vasculitis. Many different mosquitoes, including several native to North America, are competent vectors for RVFV epidemic transmission. The introduction of RVFV into North America would likely cause panic in the general population, and the effects on livestock could have a devastating economic impact. Although humoral immunity against the viral envelope proteins elicits protective immunity, there is currently no RVFV vaccine suitable for massive human vaccination programs. The present application aims to develop safe, live RVFV vaccines by modifying the attenuated MP-12 strain. Using reverse genetics and the mouse model, we will first study the genetic basis of RVFV virulence attenuation by examining the role in viral virulence of a specific amino acid in the envelope protein of wild-type RVFV. MP-12 exhibits neuroinvasiveness in young mice, and we will investigate whether this phenotype is the result of specific viral mutations accumulating during infection. Furthermore, we will generate MP-12 variants that will be less likely to produce neuroinvasive mutants. Based on the hypothesis that increasing the positive net charge of envelope proteins of MP-12 causes further attenuation of viral virulence, we will generate a series of MP-12 mutants by introducing positive charge amino acid changes into the envelope proteins, and we will test their competence for eliciting neutralizing antibodies and their virulence in mice. If these viruses demonstrate a reduced virulence and poor immunogenicity, another set of mutant viruses carrying human-codon optimized envelope protein genes will be generated and tested for virulence, immunogenicity and efficacy in protecting immunized mice from wt RVFV challenge. Codon optimization will also be tested as a method to reduce mosquito infectivity. The final product of this project will be a second generation MP-12 vaccine with increased safety, suitable for final product development and human testing. The attenuation strategies will also be applicable to other emerging bunyaviral vaccine development as well as other RNA viruses.